Uterine cancer is the 6th leading cause of cancer death amongst American women. The vast majority (98%) of uterine cancers are endometrial carcinomas (ECs), which arise from the inner epithelial lining of the uterus (the endometrium). Although the prognosis for many women with newly diagnosed endometrial cancer is good, less favorable outcomes are associated with certain disease subgroups including high-grade endometrioid ECs, late-stage endometrioid ECs, and aggressive histological subtypes such as serous EC and uterine carcinosarcoma (recently reviewed in: Urick & Bell, Nature Reviews Cancer, 2019; and Bell & Ellenson, Annual Review of Pathology: Mechanisms of Disease, 2019). Endometrioid ECs account for about 80% of newly diagnosed endometrial carcinomas. Most endometrioid ECs are detected at an early clinical stage, when surgery can be curative, but about 15% of cases are detected when they are already at an advanced stage (FIGO stage III or IV) and have metastasized beyond the uterus. Patients with advanced-stage endometrioid tumors have a relatively poor prognosis. The objectives of this project are to identify somatically mutated genes in advanced-stage endometrioid ECs and to determine whether mutations in these genes are enriched in late-stage disease compared to early-stage disease. In previous reporting periods we exome sequenced DNA from 19 late stage primary endometrioid endometrial tumors and matched non-tumor tissues and identified somatic (tumor-specific) variants in each tumor. We rigorously filtered and annotated this dataset to delineate high-confidence somatic single nucleotide variants (SNVs) and used MutSigCV to identify statistically significantly mutated genes which are by definition potential driver genes. In parallel we performed microsatellite instability testing to distinguish microsatellite stable and microsatellite unstable tumors. In the current reporting period, we extended our analysis to somatic SNVs within the 336 endometrioid EC exomes within The Cancer Genome Atlas (TCGA). Specifically, we reanalyzed the TCGA SNV data to identify candidate driver genes separately for late-stage tumors (n=66) and early-stage tumors (n=240). A comparison of these two datasets highlighted a small number of genes that are mutated more often in late-stage disease than in early-stage disease. We are currently preparing a manuscript describing our findings. Our other major objectives are to determine the molecular consequences of recurrent mutations in the FBXW7 gene in endometrial cancer, and to assess whether these molecular effects are druggable. The protein encoded by FBXW7 is a tumor suppressor and a critical component of a ubiquitin ligase complex degrades numerous substrate proteins involved in tumorigenesis. We previously discovered frequent and recurrent somatic mutations of FBXW7 in endometrial carcinomas (Nature Genetics 2012; 44:1310-5). Moreover, we found that FBXW7 mutations are particularly frequent in serous endometrial ECs. We thus hypothesized that recurrent FBXW7 mutations disrupt the proper function of the encoded protein in serous EC. In 2018, we published a manuscript reporting novel functional effects of recurrent FBXW7 mutations in serous EC (Urick and Bell, Molecular Carcinogenesis, 2018). Specifically, we showed that recurrent mutations in FBXW7 cause increased levels of phosphorylated Cyclin E1, SRC-3, c-MYC, Rictor, GSK3, P70S6, and AKT proteins in serous EC cell lines. By performing drug sensitivity assays in a serous EC cell-line that was CRISPR/Cas9-edited to knock in a series of FBXW7 mutations, we found that FBXW7-mutant serous EC cells are more sensitive to SI-2 (a SRC3 inhibitor-2) and to dinaciclib (a cyclin dependent kinase CDK inhibitor) than parental unedited cells (Molecular Carcinogenesis, 2018). Collectively the aforementioned studies shed novel insights into the functional consequences of FBXW7 mutations in serous EC. As noted previously the FBXW7 protein is a critical component of a ubiquitin ligase complex that regulates the degradation of numerous substrate proteins involved in tumorigenesis. Many of these protein substrates are transcription factors that regulate the expression of other genes. Therefore, we hypothesize that FBXW7 mutations directly and indirectly impact the levels of a multitude of proteins within the cell. Therefore, in ongoing studies our objective is to determine the consequences of FBXW7 mutations on the proteome and phosphoproteome of serous EC cells CRISPR/Cas9 edited to knock in FBXW7 mutations. These longer-term studies will extend into the next reporting period and have the potential to provide unprecedented insights into the effects of FBXW7 mutations in serous EC, one of the most clinically aggressive forms of endometrial cancer.